Morphine has been used for centuries to alleviate pain and still remains the analgesic standard to which newly developed drugs are compared. However, prolonged use of morphine, as well as other opiates, can lead to the development of tolerance and ultimately, addiction. The neuronal signaling mechanisms by which options mediate antinociception through their G protein-coupled receptors is not clearly understood. Likewise, the process of tolerance development remains to be defined at the cellular level. There is some evidence to suggest that desensitization (discontinued signaling) and/or downregulation (loss of receptors) of opioid receptors may be involved in controlling these processes. Two major classes of proteins involved in GPCR regulation are GPCR kinases (GRKs) and arrestins. Although a number of recent studies have examined the contribution of GRKs and arrestins to opioid receptor regulation in cell cultures, there have been no conclusive in vivo studies of functional significance to this date. Our laboratory has a number of transgenic mouse lines that lack different GRKs or barrestins. This report proposes the following specific aims: AIM I: To determine differences in pain perception after morphine treatment between wild type and GRK or barrestin knockout mice. AIM II: To identify whether morphine induces different degrees of tolerance between wild type and GRK or barrestin knockout mice. AIM III: To evaluate m opioid receptor function in brain and in primary neural cultures derived from wildtype and knockout mice. AIM IV: To generate a transgenic mouse that overexpresses a GRK or barrestin. By this approach we hope to identify which GRKs/arrestins are essential in morphine's actions regarding analgesia and the development of tolerance. We envision that these observations may lead to greater understanding of the mechanisms behind morphine's potent actions in vivo. Advances in this field may not only lead to potential gains in pain control, but may also lead to better treatment and possible prevention of opiate addiction.